Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

La recombinaison homologue (RH) est une réaction universelle qui assure la maintenance des génomes. Elle participe aussi à leur variabilité. De fait, dans les trois domaines du vivant elle est au centre de nombreux processus biologiques tels que la réparation des dommages à l'ADN ou encore le brassage génétique au cours de la méiose. Chez les bactéries, la RH est également impliquée dans les processus de transferts horizontaux qui favorisent les échanges génétiques entre les espèces. La transformation génétique est l'un de ces processus largement répandus parmi les bactéries. Celle-ci a spécifiquement lieu lorsque les cellules entrent dans un état physiologique particulier nommé la compétence. Comparativement aux autres types de transferts horizontaux, la transformation génétique à la caractéristique d'être entièrement contrôlée par la cellule receveuse qui dirige l'intégration d'ADN exogène capturé dans son milieu extérieur. Le but de ma thèse a été d'améliorer notre compréhension moléculaire de la voie de RH spécifique de la transformation génétique de Streptococcus pneumoniae qui permettrait à ce pathogène non seulement d'échapper aux vaccins mais également d'acquérir de nouvelles résistances aux antibiotiques. Bien qu'elle présente des variations d'un organisme à l'autre, la RH peut fondamentalement être décomposée en trois phases successives catalysées par la recombinase RecA chez les bactéries. La phase présynaptique consiste en la polymérisation de la recombinase sur l'ADN simple brin (sb) générant un nucléofilament. L'étape synaptique comprend l'appariement du nucléofilament avec celui des deux brins de la séquence homologue qui lui est complémentaire. Elle aboutit à l'apparition d'une D-loop, intermédiaire de recombinaison résultant de la jonction entre les deux molécules ADN engagées. Enfin, la phase postsynaptique constitue l'étape finale durant laquelle les jonctions entre les molécules ADN sont maturées puis clivées de manière à maintenir l'intégrité double brin du génome. L'action optimale de la recombinase au cours de ces trois étapes nécessite l'assistance de partenaires protéiques spécifiques au processus au sein duquel la réaction de RH intervient. Dans le cadre de la transformation génétique, la RMP (recombination mediator protein) DprA et la protéine de liaison à l'ADNsb SsbB sont deux partenaires de RecA spécifiquement produits par les cellules en état de compétence. Nous avons révélé in vitro que leurs actions conjuguées permettent d'améliorer l'efficacité de la RH catalysée par RecA en facilitant l'étape présynaptique de la réaction. De plus, nous avons montré que contrairement à SsbB, la protéine SsbA constitutive et essentielle ne stimule pas la RH spécifique de la transformation génétique de S. pneumoniae. Ce résultat suggère que les deux paralogues n'interviennent pas au cours des mêmes processus biologiques. La protéine RadA, ubiquitaire et très conservée parmi les bactéries, est un autre partenaire de RecA constitutivement produit par les cellules mais spécifiquement induit lors de la transformation génétique de S. pneumoniae. Par une combinaison d'approches in vivo, in vitro et structurale, nous avons mis en évidence d'une part, l'appartenance de RadA à la famille SF4 d'hélicases de type DnaB, et d'autre part son rôle clef dans la phase postsynaptique de la RH. Ce travail a permis de proposer un modèle inédit du mécanisme de migration de branches permettant la maturation des intermédiaires de recombinaison. Via son interaction avec RecA, nous proposons que RadA accède aux deux brins de l'ADN receveur de part et d'autre de la D-loop. Ainsi chargé symétriquement, RadA transloquerait de manière divergente le long des deux ADNsb dans le sens 5'-3' permettant ainsi l'ouverture du duplex receveur. L'incorporation de l'ADNsb envahissant serait ainsi facilitée par l'action de RadA tant au cours de la transformation génétique que des processus de maintenance faisant intervenir la RH / Homologous recombination (HR) is a central biological process in living organisms across all three domains of life. Crucial both for genomic maintenance and genetic plasticity, HR acts either to repair harmful DNA breaks or to produce new DNA combinations on chromosomes. In bacteria, HR is also used to exchange genetic material between different strains or species through horizontal gene transfers processes. Genetic transformation is one of those processes, widely distributed among species. Genetic transformation, occurring during a distinct physiological state called competence, is entirely directed by the recipient cell which uses exogenous DNA as a source of transferred genetic material. The goal of my PhD was to provide a molecular understanding of the specific HR pathway operating during the genetic transformation process of Streptococcus pneumoniae which enables this human pathogen to escape vaccine by capsular serotype switching as well as to acquire new antibiotics resistance genes. Although HR mechanisms vary among different organisms and cell types, the same three basic steps are conserved. During the pre-synaptic phase, the highly conserved recombinase named RecA in bacteria polymerises along ssDNA. The synaptic step proceeds through invasion of this nucleofilament into complementary duplex DNA, promoting the formation of joint molecules called D-loops. Finally, the post-synaptic phase corresponds to the maturation and clivage of thoses branched structures to preserve genomic integrity. To complete this strand exchange reaction, RecA requires the assistance of several specific partners depending on the context of the HR event. During genetic transformation, the recombination mediator protein DprA and the ssDNA binding protein SsbB are such partners specifically produced in the competence state. We have shown in vitro that their interplay with RecA improves HR efficiency by facilitating the pre-synaptic step of the reaction. Moreover, we have shown that, contrary to SsbB, the essential SsbA protein does not stimulate this specific HR reaction, suggesting that the two paralogous proteins could be implicated in different processes. The ubiquitous bacterial RadA in an other RecA partner constitutively produced by the cells and specifically induced during genetic transformation of S. pneumoniae. Using a combination of in vivo, in vitro and structural approaches, we have shown that RadA is a DnaB-like helicase implicated in the post-synaptic phase of HR. This structural and functional analysis of RadA leads to an unprecedented model of DNA branch migration acting to maturate D-loops structures. Through its interaction with RecA, RadA gains access to both strands of the recipient duplex DNA on both sides of the D-loop. Once symmetricaly loaded RadA could translocate divergently thereby unwinding the complementary duplex DNA thus facilitating the incorporation of ssDNA during genetic transformation as well as in genomic maintenance processes.

L’ADN est constamment exposé à des insultes génotoxiques endogènes et exogènes. Plusieurs mécanismes de réparations de l’ADN sont mis en œuvre pour préserver la stabilité du génome et de l’épigénome. La recombinaison homologue (RH) joue un rôle central dans la réparation des cassures double brin de l’ADN (DSBs) et le redémarrage des fourches de réplication en réponse à un stress réplicatif. Ces deux processus sont tous deux couplés à l’assemblage de la chromatine. Le facteur d’assemblage de la chromatine 1 (CAF-1) est un chaperon d’histone conservé au cours de l’évolution qui fonctionne dans le processus d’assemblage des nucléosomes couplé à la réparation de l’ADN et à la réplication, en déposant sur l’ADN les tétramères d’histones (H3-H4)2 nouvellement synthétisés. Chez la levure Schizosaccharomyces pombe, le complexe CAF-1 est constitué de trois sous-unités, Pcf1, Pcf2 et Pcf3. Il a été montré que CAF-1 agit dans l’étape de synthèse de l’ADN durant le processus de réplication dépendante de la recombinaison (RDR) et protège le désassemblage des D-loop par l’hélicase Rqh1, membre de la famille des hélicases RecQ. Dans cette étude, nous avons adressé le rôle de CAF-1 pendant la réparation de l’ADN par recombinaison homologue chez la levure Schizosaccharomyces pombe. Par l’utilisation d’approches in vivo et in vitro, nous avons validé des interactions protéines-protéines au sein d’un complexe contenant Rqh1, CAF-1, PCNA, et l’Histone H3. Nous avons montré que Rqh1 interagit avec Pcf1 et avec Pcf2 indépendamment l’un de l’autre, et que l’interaction Rqh1-Pcf1 est stimulée par des dommages à l’ADN. Nous avons mis en place une méthode d’analyse de liaison à la chromatine pour suivre l’association de CAF-1 à la chromatine en réponse aux dommages à l’ADN. Nous avons observé qu’un stress réplicatif, mais pas l’induction de cassures double brin de l’ADN, favorise l’association de CAF-1 à la chromatine. Nous avons identifié plusieurs facteurs de la RH nécessaire pour l’association de CAF-1 à la chromatine en réponse à un stress réplicatif. De plus, nous avons mis en évidence des interactions physiques entre Pcf1 et des facteurs de la recombinaison homologue, parmi lesquels RPA et Rad51. Nos données suggèrent que CAF-1 pourrait s’associer aux sites de synthèse d’ADN dépendent de la recombinaison via son interaction avec des facteurs de la RH. L’ensemble des données de cette étude contribuent à renforcer le role de CAF-1 couplé à réparation de l’ADN, et révèlent une interconnexion entre les facteurs de la RH et l’assemblage de la chromatine. / DNA is constantly exposed to both endogenous and exogenous genotoxic insults. Multiple DNA repair mechanisms are exploited to guard the genome and epigenome stability. Homologous recombination (HR) plays a major role in repairing DNA double strand breaks (DSBs) and restarting stalled replication forks under replicative stress. These two processes are both coupled to chromatin assembly. Chromatin assembly factor 1 (CAF-1) is a highly conserved histone chaperone known to function in a network of nucleosome assembly coupled to DNA repair and replication, by depositing newly synthesized histone (H3-H4)2 tetramers onto the DNA. The fission yeast CAF-1 complex consists of three subunits Pcf1, Pcf2 and Pcf3. CAF-1 has been previously reported to act at the DNA synthesis step during the process of recombination-dependent replication (RDR) and protects the D-loop from disassembly by the RecQ helicase family member, Rqh1. In this study, we addressed the role of CAF-1 during homologous-recombination-mediated DNA repair in fission yeast.Using in vivo and in vitro approaches, we validated interactions within a complex containing Rqh1, CAF-1, PCNA, and Histone H3. We showed that Rqh1 interacts with both Pcf1 and Pcf2 independently of each other, and the Pcf1-Rqh1 interaction is stimulated by DNA damage. We developed an in vivo chromatin binding assay to monitor the association of CAF-1 to the chromatin upon DNA damage. We observed that replication stress but not double strand break favors CAF-1 association to the chromatin. We identified that several HR factors are required for CAF-1 association to the chromatin upon replication stress. In support of this, we have identified physical interactions between Pcf1 and HR factors, including RPA and Rad51. Our data suggest that CAF-1 would associate with the site of recombination-dependent DNA synthesis through physical interactions with HR factors. Put together, this work contributes to strengthening the role of CAF-1 coupled to DNA repair, and reveals the crosstalk between HR factors and chromatin assembly.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The LuxR protein family is a class of quorum-sensing regulated bacterial transcription factors that alter gene expression as a function of ligand detection. This coincides with a high population density and/or a low rate of signal ligand diffusion. The majority of LuxR proteins are activated only in the presence of the signal ligand, an acyl-homoserine lactone (AHL). EsaR, from the corn pathogen Pantoea stewartii, represents a subset of LuxR homologues that are active in the absence of AHL and deactivated by its presence. The mechanism by which EsaR responds to AHL in a manner opposite to that of the majority of LuxR homologues remains elusive. Unlike the majority of LuxR homologues, which require AHL for purification, EsaR can be purified and biochemically investigated in the absence and presence of AHL. This work sought to answer questions regarding the structure-function relationship of the LuxR homologue, EsaR. Fluorescence anisotropy was used to determine the relative DNA-binding affinity of wild type EsaR and three AHL-independent EsaR variants in the presence and absence of AHL. This enabled for quantitative analysis of the relative binding affinities of these AHL-independent variants for the EsaR binding site, the esa box. The results demonstrate that one AHL-independent EsaR variant has a slightly higher affinity for the esa box in the presence, rather than the absence of AHL. The affinity of the other two for the DNA is not impacted by AHL, potentially due to an inability to transduce the signal of ligand detection to the DNA binding domain. Constructs containing only the EsaR N-terminal domain (NTD) were also developed. These constructs circumvented solubility issues associated with the full-length protein, allowing for additional biochemical analysis. It was determined that the EsaR NTD alone is sufficient for multimerization and ligand binding. Additionally, preliminary X-ray crystallography efforts have established some of the early parameters required to solve the crystal structure of the EsaR ligand binding domain in both the presence and absence of AHL. If pursued, these structures would be the first solved of a LuxR homologue ligand binding domain in both the presence and absence of the native AHL, potentially demonstrating the conformational change that occurs as a result of ligand binding. Collectively, these findings have established some of the groundwork required to resolve the question of what sort of conformational changes occur in EsaR as a result of ligand binding. / Master of Science

The LuxR family of proteins serves as quorum-sensing transcriptional regulators in proteobacteria. At high population densities, a small acyl-homoserine lactone (AHL) molecule, produced by a LuxI homologue, accumulates in the environment. The LuxR proteins bind to their respective AHL when the ligand accumulates to sufficient levels. Once bound to AHL, the holoproteins usually become functional as transcriptional activators. However, there is a subset of LuxR homologues, the EsaR subfamily, which is active without the AHL ligand and becomes inactivated once bound to it. EsaR is the best understood member of this subfamily. It controls virulence in the corn pathogen Pantoea stewartii ssp. stewartii. Solubility issues have previously limited structural studies of LuxR homologues as the proteins could not be purified without the AHL ligand. A soluble recombinant EsaR protein, HMGE, is biologically active and can be purified in the absence and presence of AHL, unlike most other LuxR homologues. Using HMGE, amino acid substitutions and Förster resonance energy transfer (FRET), experimental methods were designed for determining the dimerization interface of EsaR and for testing the hypothesis that EsaR undergoes a conformational shift when presented with the AHL ligand. To identify residues of the dimerization interface, heterodimerization assays were designed, involving either coexpression or coincubation of wild-type EsaR and variant HMGE proteins. In this assay, the inability of the proteins to copurify by nickel affinity chromatography would indicate that the modified residue(s) are important for dimerization of EsaR. To determine the conformational change that EsaR undergoes when bound to the AHL ligand, a FRET assay was developed to estimate the distances between amino acid residues in the absence and presence of AHL. Future work will have to include a few modifications to the methods and/or control experiments. This study provides the basis upon which the present methods can be further developed and later used for structural studies of EsaR. / Master of Science

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

The Disrupted-In-Schizophrenia 1 (DISC1) gene is one of the most established risk genes for psychiatric illness currently being studied, having originally been identified as being directly disrupted by a balanced chromosomal translocation that cosegregates with schizophrenia and other major mental illness a large Scottish family. The DISC1 protein is believed to act as a molecular scaffold within the cell, binding to a large number of other proteins. Three of these protein interactors, Phosphodiesterase 4B (PDE4B), Nuclear Distribution Factor E (Aspergillus nidulans)-homologue 1 (NDE1) and NDE-Like 1 (NDEL1) all have evidence implicating them as schizophrenia-related proteins in their own right. NDE1 and NDEL1 are highly similar proteins which are known to play cellular roles including microtubule function and mitosis. Their orthologues have also been shown to be important in neurodevelopment within the mouse brain. To date, most work in the literature has investigated NDEL1, with few focusing on NDE1. In the thesis, I first seek to establish a basic biology for NDE1 by the identification of splice variants expressed in the brain, establishing cellular localisation patterns within the cell and investigating NDE1 multimerisation. The relationship between NDE1 and NDEL1 is also investigated, with the two being found to form complexes together and to have partially over-lapping expression patterns within the cell. That NDE1 and DISC1 directly interact is confirmed. The relationship between NDE1 and PDE4B is then investigated, with the two proteins found to complex within the cell. Additionally, it is shown that NDE1 can be phosphorylated by protein kinase A (PKA). This kinase is cAMP dependant, and is thus indirectly regulated by the cAMP-degrading action of PDE4B protein. Attempts to map and analyse the effect of this phosphorylation on NDE1 are made.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.